The detection of molecules in samples, such as the determination of molecular interactions, in particular protein-protein interactions, is key in many biological fields, particularly cancer biology.
For example, understanding and measuring the molecular diversity underpinning tumour heterogeneity has recently become a major concern, as failure to appreciate this is thought to be one of the reasons for therapeutic failure, particularly in advanced solid tumours.
Intra-tumoural genetic heterogeneity is increasingly well documented in solid cancers, however there is little as yet understood about functional molecular heterogeneity of tumours. The appreciation of this functional heterogeneity has far reaching implications for the development of personalized medicine and improving tumour biopsy methodologies for predictive biomarkers.
For example, breast cancer is heterogeneous at both the histological and molecular levels. A better understanding of breast tumour heterogeneity at the molecular level among a large cohort of patients will help to identify the subtypes to predict outcome, patient response to chemotherapy or targeted therapy.
The assessment of molecular heterogeneity at the protein level has not been extensively reported, in part due to a lack of technologies that can accurately perform this task directly on histological samples. Formalin fixed paraffin embedded (FFPE) tissue sample preparation is the current standard technique by which outpatient and surgical biopsies are processed. Protein biomarkers and their post-translational modifications can be preserved in FFPE archived tumour samples and hence these lend themselves to the accurate quantification of onco-protein functional status and heterogeneity provided the analytical tools and processes are appropriate.
Accurate measurement of the functional status of onco-proteins and tumour heterogeneity at the molecular level may aid the ability to identify subtypes in order to predict outcome, patient response to chemotherapy or targeted therapy.
One well characterized onco-protein, Akt/PKB (protein kinase B) is a member of the AGC family of protein serine/threonine kinases and contains an N-terminal pleckstrin homology (PH) domain which interacts with Ptdlns(3,4)P2 and Ptdlns(3,4,5)P3. In cancer, Akt plays a central role in cell proliferation and survival, glucose metabolism, genome stability, and neo-vascularization. Akt also contributes to tumour invasion and metastatic spread by induction of epithelial-mesenchymal transition (EMT). Dysregulation of Akt signaling is considered to be a hallmark of many human cancers. In breast cancer, Akt activation occurs in high-grade cases and is correlated with advanced disease, poor prognosis, reduced patient survival, and tumour radioresistance. Various mechanisms contribute to activation of the Akt pathway in human tumours, including disruption of PTEN, up-regulation of phosphoinositide 3-kinase (PI3K) and down-regulation of mTOR (mammalian target of rapamycin). The Akt pathway is also activated by numerous growth factors and cytokines through their cognate receptors. Stimulation of the epidermal growth factor receptor (EGFR) by epidermal growth factor (EGF) leads to activation of Akt in a PI3K-dependent manner. Since Akt activation is both an early event in tumour progression and also a characteristic of many advanced carcinomas, it may represent a useful therapeutic target in both adjuvant and metastatic settings. Therefore, the accurate quantification of its activation state as well as its molecular heterogeneity in patient samples would be highly informative.
At present, research involving detection (and quantification) of molecules, such as endogenous proteins, including onco-proteins in fixed tumour tissue samples, faces several important challenges:
i) the accurate quantification of post-translational modifications, such as phosphorylations, and of protein-protein interactions, such as the interaction of proteins in a complex;
ii) the simultaneous localization of protein in a preserved tissue architecture;
Immunohistochemistry (IHC) is the most readily available method to assess activation of intracellular proteins, such as Akt. However, owing to it being intensity-based, it has several limitations such as lack of standardized scoring, subjectivity in the interpretation of labeled samples, and absence of precise quantification. In addition, it is a “one-site” assay that limits specificity.
FRET makes it possible to measure the interactions (association or dissociation) between two molecules, such as proteins, in close proximity (<10 nm) that are labeled with a pair of fluorescence dyes. A donor fluorescent dye has shorter excitation/emission wavelengths, that excites an acceptor fluorescence dye if the excitation spectrum of the acceptor overlaps with the emission spectrum of the donor. Since the efficiency of energy transfer reduces by the sixth order of magnitude of the distance between the fluorescent dyes, efficient energy transfer generally only occurs between fluorescent pairs that are less than 10 nm apart. Therefore, this approach can be used to detect close spatial association of molecules by labelling them with fluorescent dyes. This approach can also be used to measure protein complex formation as well as conformational changes in molecules, such as conformational and post-translational modification states of individual proteins.
A key limitation of use of FRET for detection of molecules using antibodies is the need to label a pair of antibodies with fluorophores to sufficiently high degrees to achieve adequate signal/noise ratio. Obtaining efficient FRET relies on availability of antibody pairs that are labeled with different fluorophores (either as GFP/mRFP fusion proteins or via chemical conjugation with appropriate fluorescent dyes). This process requires careful optimisation and the labelling of antibodies. Furthermore, current labelling approaches of antibodies give only sufficient signal/noise ratio for their routine use on cell lines in tissue culture—with the target molecule(s) frequently needing to be over-expressed. For tissue sections, in order for there to be sufficient binding specificity, it would be necessary to provide a low protein to dye ratio, which would result in a weak signal that is often undetectable. Hence, use of FRET on tissue sections in particular, is not routine.
In order to perform FRET on tissue sections, “coincidence FRET” or “two-site” FRET can be used. Such method involves simultaneously labelling a single protein on two distinct sites (i.e. the two target sites are on the same protein) with a FRET donor and a FRET acceptor, and detecting the FRET between them. The method has gained recent popularity due to its high specificity and its relative insensitivity to intensity artefacts. However, time resolved FRET methodologies have been limited by low sensitivity due to the requirement for fluorescently labeled primary antibodies. In particular, in order to obtain the high binding specificity, it is necessary to use a low protein to dye ratio, which results in a weak signal. Increased labelling of the antibody results in reduction of the signal to noise ratio and disrupts the antibody binding to the target region.
For example, FRET assays that quantify ectopically expressed proteins tagged with appropriate donor and acceptor pairs of fluorophores such as GFP and monomericRFP (mRFP) are well established. In these cell-based experiments, relatively elevated expression of these GFP and mRFP fusion proteins provides a high signal-to-noise ratio. However, quantifying the activation of endogenous proteins directly in cells using a coincidence FRET assays has been a challenge, partly due to the fact that in order to obtain the high binding specificity, it is necessary to use a low protein to dye ratio, which results in a weak signal.
When chromophores are conjugated to primary antibodies, high average FRET efficiencies are achieved but several limitations are encountered. Firstly, the conjugation process can result in the presence of multiple dye molecules at the antigen recognition site, with adverse consequences on antibody-antigen specificity. Secondly, the signal obtained from primary antibody-chromophore conjugates could not be amplified due to limitations in the dye-to-antibody ratio. This is particularly problematic when protein biomarkers are present in low quantities in tissue samples. Thirdly, cost becomes a limiting factor due to the large amount of primary antibodies required for antibody-chromophore conjugation. This is compounded by the fact that commercially available primary antibody-chromophore conjugates with compatible FRET pairs are often difficult to find. Making primary antibody-chromophore conjugates for use in FRET experiments is equally expensive, time-consuming in the characterization of each pairing and there is the perennial risk of loss of function associated with labelling or denaturation.
Unlabeled primary antibodies have been used in combination with chromophore-conjugated secondary antibodies. These are less preferable to primary antibody-chromophore conjugates because for efficient FRET, it is important to keep the donor and acceptor fluorophores within distances where FRET can occur i.e. less than 10 nm. With the use of primary and secondary antibodies, this can lead to increased distances of the fluorophores, thus diminishing a positive FRET signal and consequently, the signal-to-noise ratio. Such labelling methodologies give only sufficient signal/noise ratio for their routine use on cell lines—with the target molecule(s) frequently needing to be over-expressed. For tissue sections, in order for there to be sufficient binding specificity, it would be necessary to provide a low protein to dye ratio, which would result in a weak signal. This, in combination with the increased distances of the fluorophores that can occur when using primary and secondary antibodies, would result in further weakening of the signal, which often makes the signal undetectable. Hence, use of FRET on tissue sections with primary and secondary antibodies, is also not routine.
König et al. discusses the use of labeled-secondary antibodies in FRET. This article states that whole immunoglobulins as well as Fab fragments can be used as secondary antibodies with unlabeled primary antibodies. However, such methods require careful optimisation.
In other areas of biology, such as standard immunohistochemistry and other one-site methods, signal amplification methods have been used in an attempt improve the signal-to-noise ratio. One such method is tyramide signal amplification (TSA), which amplifies both chromogenic and fluorescent signals in standard immunohistochemistry methods. This methodology is based on the ability of horseradish peroxidase (HRP), in the presence of low concentrations of H2O2, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical (reactive biotinylated tyramide) that can covalently bind to electron rich moieties (such as tyrosine residues) at or near the HRP. However, the dynamics of TSA amplification and the diffusion radius of the resulting reactive species has not been well characterised. As such, TSA amplification methods are predicted to result in non-specific labelling of non-target proteins, thereby creating artefacts that will amplify indefinitely resulting in a non-specific signal.
It was also previously thought that the use of TSA amplification or similar amplification methods in a FRET method would further lead to increased distances of the fluorophores, thus diminishing a positive FRET signal and consequently, the signal-to-noise ratio.
The object of the present invention is to provide an improved method for detecting molecules, particularly protein states in cells, such as in tissue sections.